Epidemiology suggests that diabetes and hypercholesterolemia increase risks for atherosclerotic & osteoporotic diseases. The regulation of vascular calcification is poorly characterized; the LDLR -/- mouse model & human valve specimens implicate heterotopic osteogenic mechanisms. It is important to understand aortic calcification and metabolism in our aging population since pharmacotherapies implemented to promote bone formation & preserve bone mass may alter progression of calcific vasculopathy. The 3 specific aims of this proposal are: Aim 1: "To characterize the activities of prolonged dysmetabolic exposure and osteoanabolic pharmacotherapy on orthotopic (skeletal) vs. heterotopic aortic osteogenic gene expression programs, using the diabetic LDLR -/- mouse as a model." In vivo data suggest overlapping yet distinct transcription mechanisms are rate limiting in the initiation of orthotopic vs. aortic calcium deposition. We will directly assess the aortic vs. osseous responses to osteoanabolic pharmacotherapy in the presence or absence of the dysmetabolic state. PTH will be used as a relevant, prototypic osteoanabolic stimulus in the dysmetabolic LDLR -/- mouse model. Gene expression of key osteogenic transcription factors, morphogens, & matrix molecules will be quantified by fluorescence RT-PCR, & spatially resolved by in situ hybridization. Temporo-spatial deposition of aortic calcium will be quantified by image analysis of von Kossa stained sections. Aim 2: "To identify the transcriptional mechanisms that regulate aortic mesenchymal cell osteogenic gene expression, using the osteopontin (OPN) & Msx2 promoters as models for study." The goal is to identify specific DNA-protein interactions that mediate responses to dysmetabolic signals (e.g., diabetes, hyperlipidemia) that control expression of these key osteogenic genes in vascular smooth muscle cells, peri-aortic adventitial cells, and osteoblasts using (a) transfection and gel shift analyses in primary cell cultures and cell lines; and (b) cDNA cloning techniques. Aim 3: "To identify if the gene expression programs elaborated during aortic calcification in the diabetic, hyperlipidemic LDLR -/- mouse provide a molecular phenocopy of human aortic calcification via molecular analysis of calcified human valves." We will determine if human aortic valve calcification quantified by spiral CT is associated with up-regulation of specific osteoblast transcriptional regulatory programs controlled by Msx2, Dlx5, & Runx2/Cbfa1/Osf2 (osteoblast transcription factors). Gene expression will be quantified by fluorescence RT-PCR and spatially resolved by in situ hybridization. A sub-aim will validate methods for quantifying aortic valve calcium content by spiral CT for future studies of patients treated with osteoprotective agents.